This invention is related to novel cationic liposomal formulations for delivery of active agents such as oligonucleotides, proteins, or oligopeptides, oligosaccharides and chemotherapeutic agents. The invention also relates to the use of oligonucleotides, preferably having a size of xe2x89xa640 mucleotides for enhancing radiosensitivity of radiation-resistant tumors.
Radiation therapy is an important treatment modality of cancer. Such therapeutic methods of therapy include the administration of radiolabeled ligands that bind to a target site, i.e., a tumor, and the irradiation of a tumor using irradiation devices. Radiolabeled ligands used for the treatment of cancer include especially radiolabeled antibodies or radiolabeled peptides that bind to a receptor selectively expressed by a cancer cell.
Recently methods for treating cancers using radiation have improved in the fact that there exist better techniques for selectively targeting radiation to a desired site, i.e., a tumor, thereby minimizing the risk of radiation associated toxicity to normal cells and tissues. However, one prevalent problem with radiation therapy is the fact that many cancers are resistant to the cytotoxic effects of ionizing radiation.
Some researchers have theorized that resistance to irradiation may be linked to certain oncogenes, e.g., ras, raf, cot, mos, myc; growth factors (e.g., PDGF, FGF) and the phenomenon of cellular resistance to ionizing irradiation. For example, it was reported that expression of antisense C-raf-1 cDNA resulted in reduced expression of c-raf-1 gene, and provided for enhanced radiation sensitivity of radioresistant laryngeal squamous carcinoma cells (SE-20B cells) (Kasid et al, Science, 243:1354-1356 (1989)).
The use of antisense oligonucleotides for treatment of cancer has also been reported. However, previous problems associated therewith include that such oligonucleotides tend to be unstable in vivo and, therefore, may become degraded before they reach the target site, e.g., tumor cell or viral infected cell.
Attempts to increase the potency of oliogs have included the synthesis of several analogs, with modifications directed primarily to the phosphodiester backbone. For example, phosphorothioate oligonucleotides have demonstrated to exhibit enhanced resistance to nuclease digestion. Other modifications to oligonucleotides have included derivatization with lipophilic moieties such as cholesterol, and polylysine to enhance cellular uptake. Alternatively, the polyanionic nature of the molecule has been eliminated in methylphosphonate analogs.
Another reported approach has involved the use of cationic liposomes to enhance delivery. Bennet et al., Mol Pharmacol, 4:1023-1033 (1992).Zelphati et al, J. Lipsome Res., 7(1):31-49 (1997); Thierry et al, Biochem. Biophys. Res. Comm., 190(3):952-960 (1993). It is widely accepted that cationic liposomes must contain enough charge to neutralize the negatively charged oligonucleotides as well as providing enough residual positive charge to the complex to facilitate interaction with a negatively charged cell surface. (Litzinger et al, J. Liposome Research, 7(1):51-61 (1997)). However, problems associated with previous cationic liposomal delivery systems similarly include serum-instability, undesirable biodistribution, and target-non-specificity which hinder their use for efficient nucleic acid delivery in vivo.
Thus, improved liposomal delivery systems, especially for delivery of bioactive agents such as oligonucleotides which are stable and result in delivery of an encapsulated active agent to an active site would be highly beneficial. Additionally, improved methods for treating cancers that are radiation-resistant would be beneficial.
It is an object of the invention to provide novel cationic liposomal delivery systems, especially for delivery of oligonucleotides to target cells.
It is a more specific object of the invention to provide cationic liposomes having enhanced serum stability and targeting capability that comprise dimethyldioctadecyl ammonium bromide (DDAB), phosphatidylcholine (PC), and cholesterol (CHOL).
It is another specific object of the invention to provide cationic liposomes that comprise the cationic liposome 1,2-dimyristoyl-3-trimethyl ammonium propane (DMTAP); phosphatidylcholine (PC), and cholesterol (CHOL), and having encapsulated therein a desired active agent, preferably an oligonucleotide.
It is another object of the invention to provide cationic liposomes comprising at least one cationic lipid selected from: 1,2-dioleoyl-3-trimethyl ammonium propane (DOTAP), N-(2,3-(dioleoyloxy)propyl)-N,N,N-trimethyl ammonium chloride, or 1-[2-(9(Z)-octadecenoyloxy)-ethyl]-2-(8(Z)heptadecenyl)-3-(2-hydroxyethyl)-imidazolinium chloride) phosphatidylcholine (PC) and cholesterol (CHOL).
It is an even more specific object of the invention to provide novel cationic liposomes comprising 1,2-dimyristoyl-3-trimethyl ammonium propane (DMTAP); phosphatidylcholine (PC), and cholesterol, wherein the respective molar ratios range from 0.5 to 1.4; 2.0 to 4.0; and 0.5 to 2.5; and more preferably 0.75 to 1.25; 3.0 to 4.0; and 1.25; 3.0 to 4.0; and 1.0 to 2.0; and most preferably about 1:3.2:1.6.
It is another specific object of the invention to provide cationic liposomes comprising dimethyldioctadecyl ammonium bromide (DDAB), phosphatidylcholine (PC) and cholesterol, wherein the respective molar ratios are 0.5 to 1.5; 2.0 to 4.0, and 0.5 to 2.5; more preferably 0.75 to 1.25; 3.0 to 4.0; and 1.0 to 2.0; and most preferably about 1:3.2:1.6.
It is another specific object of the invention to utilize cationic liposomes comprising at least one cationic lipid selected from: 1,2-dimyristoyl-3-trimethyl ammonium propane (DMTAP), dimethyldioctadecyl ammonium bromide (DDAB), 1,2-dioleoyl-3-trimethyl ammonium propane, (DOTAP) N-[2,3-(dioleoyloxy)propyl)-N,N,N-trimethyl ammonium chloride and 1-[2-(9-(Z)-octadecenoyloxy)-ethyl]-2-(8(Z)heptadencenyl)-3-(2-hydroxyethyl)-imidazolium chlorine; phosphatidylcholine and cholesterol, wherein the molar ratio of total cationic lipid, phosphatidylcholine, and cholesterol preferably ranges from 0.5 to 1.5; 2.0 to 4.0; and 0.5 to 2.5; more preferably 0.75 to 1.25; 3.0 to 4.0; and 1.0 to 2.0; and most preferably 0.8 to 1.2; 3.0 to 3.5; and 1.4 to 1.8 as a vehicle for in vivo delivery of active agents, especially oligonucleotides, proteins, peptides, chemotherapeutic agents, growth factors, cytokines, receptors, and antibodies, to a target site, e.g., a tumor or site of an infection. Most preferably, the active agent is an oligonucleotide. This oligonucleotide may be in the sense or antisense orientation relative to a gene target, e.g., an oncogene. Most preferably the oligonucleotide will be an antisense oligonucleotide.
It is an even more specific object of the invention to use the subject cationic liposomes for delivery of active agents, e.g., oligonucleotides, to solid tumors and cancers including head and neck cancer, prostate cancer, pancreatic cancer, breast cancer, lung cancer, kidney cancer, ovarian cancer, brain cancer, esophageal cancer, bladder cancer, liver cancer, colon cancer, penile cancer, B and T cell lymphomas, testicular cancer, bone cancer, and hematologic cancers.
It is another object of the invention to administer antisense oligonucleotides corresponding to portions of oncogenes preferably selected from the group consisting of ras, raf, cot, mos, myc, preferably c-raf-1, or a growth factor PDGF, FGF, EGF), as an adjunct to radiotherapy, in order to radiosensitize cancer cells to the effects of radiation. Preferably, such oligonucleotides will be administered using a cationic liposomal delivery system, more preferably the cationic liposomal delivery systems discussed supra. The bases comprised in said oligonucleotide may be modified or unmodified, and the size of such oligonucleotides will preferably range from 8 to 100 nucleotides; more preferably 12 to 60 nucleotides, most preferably from 15 to 40, or 15 to 25 nucleotides.
It is an even more specific object of the invention to administer oligonucleotides comprising 5xe2x80x2-GTG-CTCCATTGATGC-3xe2x80x2 (SEQ ID NO: 1) and/or 5xe2x80x2-CCTGTATGTGCTCCATT-GATGCAGC-3xe2x80x2 (SEQ ID NO: 2), preferably encapsulated in a cationic liposome, wherein the bases of said oligonucleotides may be modified or unmodified, as an adjunct to radiotherapy.